The objective of this proposal is to understand the mechanism of chromatin assembly, especially regarding how this process is coupled to DNA synthesis. Proper chromatin assembly is important for the transfer of epigenetic marks from mother to daughter cells and also for maintaining the integrity of the organism's genome. This proposal will focus on determining the mechanism of chromatin assembly facilitated by the histone H3/H4 chaperone Chromatin Assembly Factor 1 (CAF-1) using a combination of reconstituted DNA replication assays as well as biochemical and biophysical characterization of the CAF-1 complex. The stoichiometry of CAF-1 mediated histone incorporation will be investigated during replication-coupled chromatin assembly. Replication coupled assays will be developed to identify accessory proteins and reaction intermediates during replication coupled chromatin assembly. Additionally non-replication based Asf1-dependent CAF-1 chromatin assembly will be investigated using biochemical assays.